WO1985002258A1 - Novel immunoassays and kits for use therein - Google Patents

Novel immunoassays and kits for use therein Download PDF

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Publication number
WO1985002258A1
WO1985002258A1 PCT/US1984/001888 US8401888W WO8502258A1 WO 1985002258 A1 WO1985002258 A1 WO 1985002258A1 US 8401888 W US8401888 W US 8401888W WO 8502258 A1 WO8502258 A1 WO 8502258A1
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WIPO (PCT)
Prior art keywords
ab
ag
sc
antibody
type
Prior art date
Application number
PCT/US1984/001888
Other languages
French (fr)
Inventor
Linda Katherine Cragle
Paul C. Harris
Shih-Yun Lee
Ker Kong Tung
Morton Alan Vodian
Original Assignee
Beckman Instruments, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US553,219 priority Critical
Priority to US06/553,219 priority patent/US4595661A/en
Application filed by Beckman Instruments, Inc. filed Critical Beckman Instruments, Inc.
Publication of WO1985002258A1 publication Critical patent/WO1985002258A1/en

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306Solid-phase reaction mechanisms
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/81Packaged device or kit
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/962Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/975Kit
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/805Optical property
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/807Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808Automated or kit
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/815Test for named compound or class of compounds
    • Y10S436/817Steroids or hormones
    • Y10S436/818Human chorionic gonadotropin
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/819Multifunctional antigen or antibody

Abstract

A improved immunoassay for assaying an antigenic substance (Ag) in a fluid. The immunoassay is the type which comprises contacting the fluid with at least one first entity selected from a group consisting of an antibody (Ab) to the Ag, a soluble, labeled antibody (L-Aba) to the Ag, and an antibody (Abb) to the Ag bound to a solid support (SC). The immunoassay is characterized in that the fluid is contacted with at least one additional entity selected from a group consisting of at least one different type of soluble, labeled antibody (L-Abc) to the Ag, at least one different type of antibody (Abd) bound to a solid carrier (SC1), and at least one different type of antibody (Abe) to the Ag. The SC1 is selected from a group consisting of SC, at least one different solid carrier (SC2), and mixtures thereof (SC and SC2). Each type of L-Abc, Abd-SC1, and Abe has a lower average affinity constant (K) for Ag than each respective K of L-Aba, Abb-SC and Abe; and the additional entity is present in an amount sufficient to avoid a hook effect. Also, an improved reagent of the type comprising at least one first entity selected from a group consisting of L-Aba, Abb-SC, and Ab. The reagent is characterized in that it further comprises at least one additional entity selected from a group consisting of at least one different type of L-Abc and at least one different type Abd-SC1, and at least one different type of Abe. Ab, L-Aba, Abb-SC, L-Abc, Abd-SC1, and Abe are as defined above.

Description

NOVEL IMMUNOASSAYS AND KITS FOR USS THEREIN

Background of the Invention

1. Field of the Invention

This invention relates to immunoassays and to kits for use therein.

2» Description of the Prior Art

Numberous types of immunoassays presently exist. To illustrate, one- and two-step sandwich immuno¬ assays for antigens which combine to at least two anti¬ bodies are known (1-4). For example, U.S. Patent 4,244,940 (2) discloses a sandwich immunoassay method in which a sample containing an antigen (Ag) to be deter¬ mined, a labeled antibody (L-Aba) to the Ag, an antibody (Abtø) for the Ag bound "to a solid-phase support (SC) are brought together in a single incubation mode or step in an aqueous medium to form a substantially stable suspen¬ sion and produce a two-phase system. The solid phase portion of this two-phase system contains the Ab^-SC, a portion of which has become bound to the Ag which in turn has become bound to a portion of the L-Aba (represented as L-Abg-Ag-Abj-j-SC) . The liquid phase portion of the two-phase systems contains the unbound portion of the L- Aba. The solid and liquid phases are separated and either phase analyzed for the L-Aba, the concentration of which is a function of the concentration of Ag- in the sample. U.S. Patent 4,244,940 (2) teaches that this two- site immunoassay employing-.a single incubation mode or step provides significant advantages over assay pro¬ cedures involving more than one incubation by simplify¬ ing, shortening, and rendering more convenient the per¬ formance of the assay. Furthermore, U.S. Patent

OMPI 4,244,940 (2) teaches that this improvement in assay procedure is accomplished while maintaining acceptable assay characteristics such as precision, specificity, and stability, in addition to being less subject to errors in timing, additions, and other manipulations.

One- and two-step sandwich immunoassays, can be used for the determination of the presence or concentra¬ tion of any Ag which can simultaneously become bound by two antibodies. This group of Ags includes, but is not limited to, placental, pituitary, calcium regulating, and adrenal medullary polypeptide hormones, protein and pro¬ tein fragments; immunoglobulins (antibodies) of various classes; viral, viral subunits, bacterial, and protozoal organisms or particles; toxins; drugs, enzymes, and tumor-asssociated antigens.

In the one- and two-step sandwich immunoassays, the antibody employed is any substance which binds the Ag with acceptable' specificity and affinity.

In one- and two-step sandwich immunoassay the L-Aba may be labeled with any of a number of known tracers. These tracers include, but are not limited to, radioactive tags, fluorescent labels, and enzyme labels.

An important component of the one- and two-step sandwich immunoassays is the SC for the Abj-,. The SC must be able to (a) be bound to the Abj.,, (b) be handled conveniently during manipulations such as pipetting and centrifuging, and (c) exhibit low nonspecific adsorption properties or be treated so that it exhibits such adsorption properties.

A pitfall in both the conventional and one-step sandwich immunoas'say has been reported (3,5-7). More

Figure imgf000004_0001
particularly, these authors have found that a potential hazard of misinterpreting the results is involved in both the conventional and one-step sandwich immunoassays because of a false negative result or "hook effect" at high concentrations of Ag.

Nomura et al. (6), who discuss this pitfall in regard to a one-step sandwich immunoassay with monoclonal antibodies for the determination of human alpha- fetόprotein (AFP), state that "theoretically but not practically, the inhibition in antigen excess region may be avoided by employing a large amount of immobilized and labeled anti-AFP." One reason that this suggestion is not practical is that the additional large amount of labeled anti-AFP suggested by Nomura et al. would result in an assay having high non-specific adsorption since non-specific adsorption is proportional to the concentra¬ tion of L-Aba employed. Another reason that this sugges¬ tion is not practical is that this additional large amount of labeled anti-AFP would 'reduce the dynamic range of the assay when an enzyme or other label requiring the use of a spectrophotometer is employed.

Miles et al. (7), who discuss this pitfall in regard to a two-step sandwich immunoassay, note that repeated washings after the first incubation does prevent the high dose hook effect. However, this technique for preventing the high dose hook effect is tedious and time consuming.

To date, there has been no convenient technique proposed for either preventing or avoiding this false negative or hook effect phenomenum in either one- or two- step sandwich immunoassays. Another immunoassay known to those skilled in the art is the direct nephelometric immunoassay. The direct nephelometric immunoassay comprises contacting a fluid containing an antigenic substance (Ag) with an antibody (Ab) to the Ag in order to form a complex Ab-Ag. A measurement of the amount of formed Ab-Ag is directly proportional to the amount of Ag in the assayed fluid.

Like the one- and two-step sandwich immuno¬ assays, "there is a potential^ hazard of misinterpreting the results because of the false negative result or "hook effect" at high concentrations of Ag.

Although in the case of the direct nephelo¬ metric immunoassay there are some electromechanical methods for safeguarding against such misinterpretation, at present there is no known chemical means for either preventing or avoiding this false negative or hook effect phenomenon in this case either.

Accordingly, it would be very desirable to have immunoassay methodologies and kits wherein this problem has been either prevented or avoided.

Summary of the Invention In accordance with the present invention, there are provided improved immunoassay methodologies and kits for use therein wherein all the advantages possessed by the prior immunoassays are maintained with the concurrent avoidance of the hook effect.

In general, the present invention encompasses an improved immunoassay for an antigenic substance (Ag) in a fluid. The immunoassay of the present invention is of the type which comprises contacting the fluid with at least one first entity selected from a group consisting of an antibody (Ab) to the Ag,'a soluble, labeled anti¬ body (L-Aba) to the Ag, and an antibody (Abj-,) to the Ag bound to a solid carrier (SC). The present immunoassay is characterized in that the fluid is contacted with at least one additional entity selected from a group con¬ sisting of at least one different type of soluble, labeled antibody (L-Abc) to the Ag, at least one different type of antibody <Abd) to the Ag bound to a solid carrier (SC!), and at least one different type of antibody (Abe) to the Ag.

SC]_ is selected from a group consisting of SC, one or more different solid carriers (SC2)r and mixtures thereof (SC and SC2).

Each of the additional entities has an average affinity constant (K) for the Ag lower than the K of its corresponding first entity for the Ag. In addition, the additional entity is present in an amount sufficient. to avoid the hook effect.

More particularly, the present invention encompasses an improved one-step sandwich immunoassay. This one-step sandwich immunoassay of the present inven¬ tion is of the type which comprises:

(a) contacting the fluid with (i) the L-Aba and (ii) the A f-, bound to the SC to form an insoluble complex (L-Aba-Ag-Abj-,-SC) ;

(b) separating the L-Aba-Ag-Abtø-SC from the fluid and unreacted L-Aba; and

Figure imgf000007_0001
(c) measuring either the amount of L-Aba associated with the L-Aba-Ag-Abb-SC or the amount of unreacted L-Aba.

The improved one-step sandwich immunoassay of this inven¬ tion is characterized in that the fluid is contacted with at least one additional entity selected from the group consisting of one or more different type of L-Abc and one or more different type of Abd bound to the SC^;

wherein:

(i) each different type of L-Abc and Abd-SC;]_ has a lower K for Ag than each respective K of L-Aba and Abj-j-SC or Ag; and

(ii) the additional entity is present in an amount sufficient to avoid the hook effect.

More particularly, the present invention also encompasses an improved two-step sandwich immunoassay. This immunoassay is of the type which comprises:

(a) contacting the fluid containing the Ag with the Abj-j-SC to form an insoluble complex (Ag-Abb- SC);

(b) contacting the Ag-Ab-SC with the L-Aba to form an extended insoluble complex (L-Aba-Ag-Abb- SC) ;

(c) separating the L-Aba-Ag-Abb-SC from unreacted L-Aba; and

(d) measuring either the amount of L-Aba associated with the L-Aba-Ag-Abb-SC or the amount of unreacted L-Aba.

'* The improved two-step sandwich immunoassay of this inven¬ tion is characterized in that in step..(b) the Ag-Ab^-SC is contacted with one or more different type of L-Abc, ■ wherein each different type of L-Abc has a lower K for Ag than the K of L-Ab= and L-Ab„ is present in an amount sufficient to avoid the hook effect.

More particularly, the present invention also encompasses an improved direct nephelometric immuno¬ assay. This immunoassay is of the type which comprises:

(a) contacting the fluid with Ab in order to form a complex Ab-Ag; and

(b) measuring the amount of formed Ab-Ag.

The improved direct nephelometric immunoassay of this invention is characterized in that the fluid is contacted with at least one additional type of antibody Abe. Each type of Abe has a K for the Ag lower than the K of the Ab for the Ag and is present in an amount sufficient to avoid the hook effect.

Also within the general scope of this invention is an improved reagent. This improved reagent is of the type comprising at least one first entity selected from a group consisting of Ab, L-Aba, and Ab^-SC. The reagent of the present invention is characterized in that it further comprises at least one additional entity selected from a group consisting of at least one different type of L-Abc, at least one different type of Ab^-SC^, and at least one different type of Abe. The additional entity is present in an amount sufficient to avoid a hook effect when the reagent is employed in an immunoassay. In the case of one- and two- step sandwich immunoassays, the reagent is of the type comprising L~Aba and Abb-SC. The improved reagent of the present inven¬ tion is characterized in that it further comprises at least one additional entity selected from a group con¬ sisting of one or more different types of L-Abc and one or more different types of Abd bound to SC^. The addi¬ tional entity is present in an amount sufficient to avoid the hook effect when the reagent is employed in the immunoassay.

In the case of direct nephelometric immuno¬ assays, the reagent is of the type comprising Ab. The improved reagent of the present invention further com¬ prises at least one different type of Abe in an amount sufficient to avoid the hook effect when employed in a direct nephelometric assay.

Description of the Preferred Embodiments The average affinity constant (K) for each different type of L-Abc, Ab^-SC^, and Abe for Ag' ust be lower than each respective K of L-Aba, Ab^-SC and Ab for Ag. This restriction assures that the former entities compete at most only insignificantly with the latter entities in the dynamic range. In order to achieve this result, it is preferred that each K of each different type of L-Abc, Ab^-SC^ , and Abe for Ag be at least 5, more preferably at least 10 times lower than each respective K of L-Aba, Abb-SC, and Ab for Ag.

In addition, the further one wishes to extend the avoidance of the hook effect, the larger the differ¬ ence must be between (a) the K of L-Aba and the K of L-Abc, (b) the K of Abb-SC and the K of Abd-SC]_, and (c) the K of Ab and the K of Abe .

CMPI When more than one type of L-Abc, Abd-SC;]_, or Abe is employed, each different type preferably has a different K.

The amount of L-Abc employed can be any amount sufficient to extend the commencement of the hook effect and up to that which gives unsatisfactory non-specific adsorption. More particularly, this amount can range from about 0.01 to about lμg per ml of reagent.

The amount of Abd employed can be any amount sufficient to extend the commencement of the hook effect and up to the amount required to saturate SC]_. More particularly, this amount can range from 0.1 to about 10 μg per test.

The amount of Abe employed can be any amount sufficient to extent the commencement of the hook effect.

In order to obtain the advantages of the pres¬ ent invention in a one- or two-step sandwich immunoassay, one need only employ either L-Abc or Abd-SC]_. Since it is easier to adjust the labeled antibody concentration and since it is also easier to adjust the ratio of L-Aba to L-Abc, it is preferred to only employ L-Abc as the additional entity.

Aba, bb, Abc, bd, and Abe are each indepen¬ dently selected from a group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof. For example, in one- and two-step sandwich immunoassays, (a) Aba, Abb, Abc, and Abd can be mono¬ clonal antibodies or can be polyclonal antibodies; or (b) Aba and Abc can be both monoclonal antibodies and Ab^ and Abd can be both polyclonal antibodies; or (c) Aba and Abc can be both polyclonal antibodies and Abb and Abd are both monoclonal antibodies. In one- and two-step sand¬ wich immunoassays, Abc and Abd are preferably monoclonal antibodies.

The following examples are provided for pur¬ poses of further illustration only and are not intended to be limitations on the disclosed invention.

Example 1 IgE Enzyme Immuno Assay (Prior Art)

Materials

Polystyrene bead coated with a monoclonal antibody directed against a specific IgE site (Abb-SC).

Horse radish peroxidase-labeled antibody directed against different IgE sites (L-Aba) and having a K of about 2 x 1Q10.

o-Phenylenediamine

Hydrogen peroxide

Stopping solution

IgE Standards 0, 10, 25, 75, 200, and 400 IU/ml

Substrate buffer

Protocol

1. Allow all components and samples to equilibrate to room temperature and mix well prior to use.

2. Add 20 μl standard (including zero dose or sample to each test tube. 3. .Add 300 μl diluted conjugate to each tube.

4. Mix by gently shaking tube rack. Tap rack to displace air bubbles.

5. Remove bead basket from container with forceps. Hold over container until drained and place on inverted cap. Place one bead in each tube.

6. Gently shake tube rack to mix. Tap rack to remove air bubbles if necessary.

7. Incubate the tubes in a 37 ± 1° C. water bath for 30 minutes.

8. Prepare the substrate solution during the last five minutes of the incubation. Use plastic

, forceps to handle tablets.

9. Wash beads by filling each tube with approxi¬ mately 3 ml distilled water and aspirate. Repeat two more times.

10. At timed intervals, add 300 μl of enzyme sub¬ strate solution to each tube.

11. Shake test tube. ack to ensure mixing and cover tubes to exclude light.

12. Incubate 30 ± 1 minute at 20° C. to 25° C.

13. At timed intervals, add 1 ml Stopping Solution to each tube. Add in the same sequence and with the same timing as used for substrate addition. Mix by gentle shaking.

OMPI 14. Zero the spectrophotometer against distilled water and measure the absorbance of samples and standards at 492 nm. Calculate corrected absorbances for each standard and sample by subtracting the absorbance of the reagent blank (zero dose) .

Calculations

1. On semi-logarithmic graph paper, label the linear axis with absorbance (0 to 2.00A) and label the logarithmic axis with the concentra¬ tion of the standards (10 to 400 IU/ml).

2. Prepare the standard curve by plotting the average corrected absorbances of the standards, and connecting' the points with straight lines.

3.. Use the corrected absorbances of the samples to interpolate sample concentrations from the standard curve.

The results of this experiment are set forth in

Table I.

Examole 2

IgE Enzyme Immunoassay (Present Invention)

The materials, protocol, and calculations set forth in Example 1 were employed with one modification. The sole modifiction consisted of -the use of one addi¬ tional type of horse radish peroxide-labeled antibody directed against different IgE sites (L-Abc) and having a K of about 3 x 10 . The results of this experiment are also set forth in Table I. TABLE I

Absorbance

Prior Art Present Invention

IgE , IU/ml (Example 1) (Example 2)

0 0.089 0.126

10 0.161 0.186

25 0.282 0.323

75 0.614 0.634

200 1.069 1.099

400 1.396 1.592

1,r000 1.916 2.226

4, ,000 1.994 2.808

8, ,000 1.551 2.718

16, 000 1.387 3.018

40, 000 1.013 2.826

The IgE assays of Examples 1 and 2 have a dynamic range of. from 0 to 400 IU IgE per ml. Accor¬ dingly, the data set forth in Table I indicate that at IgE concentrations greater than 8000 IU/ml, one would obtain a false negative result with the prior art pro¬ cedure because of the presence of the hook effect. In contrast., with the procedure and kit of the present invention, the hook effect is avoided for IgE concentra¬ tions of at least 40,000 IU/ml. As the data of Table I also show, this avoidance is accomplished without any significant elevation of signal in the dynamic range.

Example 3 HCG Enzyme Immuno Assay (Prior Art)

Materials

Polystyrene bead coated with a monoclonal antibody directed against a specific HCG site (Abb-SC).

.XSX-EA

OMPI , WIPO Horse radish peroxidase-l.abeled antibody directed against different HCG sites (L-Aba) and having a K of about 8 x 1010.

o-Phenylenediamine

Hydrogen peroxide

Stopping solution

HCG Standards 0, 10, 25, 75, 200, and 400 IU/ml

Substrate buffer

Protocol

1. Allow all components and samples to equilibrate to room temperature and mix well prior to use.

2. Add 200 μl standard ('including zero dose or sample to each test tube.

3. Add 200 μl diluted conjugate to each tube.

4. Mix by gently shaking tube rack. Tap rack to displace air bubbles.

5. Remove bead basket from container with for¬ ceps. Hold over container until drained and place on inverted cap. Place one bead in each tube.

6. Gently shake tube rack to mix. Tap rack to remove air bubbles if necessary.

7. Incubate the tubes on a clinical rotor at 190 ± 10 RPM for 45 minutes at 20° C. to 25° C.

OMPI 8. Prepare the substrate solution during the last five minutes of the incubation. Use plastic forceps to handle tablets.

9. Wash beads by filling each tube with approxi¬ mately 3 ml distilled water and aspirate. Repeat two .more times.

10. At timed intervals, add 300 μl of enzyme sub¬ strate solution to each tub'e.

11. Shake test tube rack to ensure mixing and cover tubes to exclude light.

12. Incubate 30 ± 1 minute at 20° C. to 25° C.

13. At timed intervals, add 1 ml Stopping Solution to each tube. .Add in the same sequence and with the same timing as used for. substrate addition. Mix by gentle shaking.

14. Zero the spectrophotometer against distilled water and measure the absorbance of samples and standards at 492 nm. Calculate corrected absorbances for each standard and sample by subtracting the absorbance of the reagent blank (zero dose) .

Calculations

1. On semi-logarithmic graph paper, label the linear axis with absorbance (0 to 2.00A) and label the logarithmic axis with the concentra¬ tion of the standards (1 to 100 mlU/ l). 2. Prepare the standard curve by plotting the average corrected absorbances of the standards, and connecting the points with straight lines.

3. Use the corrected absorbances of the samples to interpolate sample concentrations from-the standard curve.

The results of this experiment are set forth in Table II.

Example 4 HCG Enzyme I muno Assay (Present Invention)

The materials, protocol, and calculations set forth in Example 3 were employed with one modification. The sole modification consisted of the use of one addi¬ tional type of horse radish peroxide-labeled antibody directed against different HCG sites (L-Abc) and having a K of about 2 x 10 . The results pf this experiment are also set forth in Table II.

TABLE II t Ab;sorbanci a

HCG, Prior Art Present Invention mlU/ml (E:xample 3) (E:xample 4)

0 0.090 0.092

1 0.101 0.108

2.5 0.130 0.132

5 0.173 0.188

10 0.290 0.284

25 0.546 0.557

50 0.952 0.932

100 1.620 1.592

1000 3.82 3.855

10000 1.64 5.514

64880 0.66 4.37

28000 0.42 3.38 The HCG assays of Eamples 3 and 4 have a dynamic range from 0 to 100 lU HCG per ml. Accordingly, the data set forth in Table II indicate that at HCG con¬ centrations greater than 10-000 mlU/ml, one would obtain a false negative result with the prior art procedure because of the presence of the hook effect. In contrast, with the procedure and kit of the present invention, the hook effect is avoided for HCG concentrations of at least 128,000 mlU/ml. As the data of Table II also show, this avoidance is accomplished without any significant eleva¬ tion of signal in the dynamic range.

Based on this disclosure, many other modifica¬ tions and ramifications will naturally suggest themselves to those skilled in the art. These are intended to be comprehended as within the scope of this invention.

OMPI Bibliography

1. U.S. 4,376,110

2. U.S. 4,244,940

3. Miles et al.. Analytical Biochemistry, 61:209-224 (1974)

4. Uotila et al. , Journal of Immunological Methods, _42_:11-15 (1981)

5. Ng et al., Clin. Chem. , _29_(6) :1109-113 (1953)

6. Nomura et al.. Journal of Immunological Methods, _5 :13-17 (1983)

7. Miles et al.. Symposium on Radioimmunoassay and Related Procedures in Clinical Medicine and Research, International Atomic Energy Agency, Vienna, Austria (1973) pp. 149-164.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. An improved immunoassay for an antigenic substance (Ag) in a fluid, said immunoassay being of the type comprising contacting said fluid with at least one first entity selected from a group consisting of (a) an antibody (Ab) to said Ag, (b) soluble, labeled antibody (L-Aba) to said Ag, and (c) antibody (Abb) to said Ag bound to a solid carrier (SC), character¬ ized in that said fluid is contacted with at least one additional entity selected from a group consist¬ ing of at least one different type of soluble labeled antibody (L-Abc), at least one different type of antibody (Abd) to said Ag bound to a solid carrier (SC]_), and at least one different type of antibody (Abe) to said Ag,
wherein:
(i) each of said additional entities has an average affinity constant (K) for said Ag lower than the K of its corresponding first entity for said Ag; (ii) said additional entity is present in an amount sufficient to avoid a hook effect; and (iii) said SC]_ is selected from a group consist¬ ing of said SC, at least one different solid carrier (SC2), and mixtures threof (SC and SC2) •
2. The improved immunoassay of claim 1 wherein said immunoassay is a one-step sandwich immunoassay of the type comprising:
(a) contacting said fluid with (i) said soluble, labeled antibody (L-Aba) to said Ag and (ii) said antibody (Abb) to said Ag bound to said solid carrier (SC) to form an insoluble complex
(L-Aba-Ag-Abb-SC) ; (b) separating said L-Aba-Ag-Abb-SC from said fluid and unreacted L-Aba; and . (c) measuring either the amount of L-Aba associated with said L-Aba-Ag-Abb-SC or the amount of unreacted L-Aba; and is characterized in that said fluid is contacted with at least one additional entity selected from said group consisting of at least one different type of soluble, labeled antibody (L-Abc) to said Ag and at least one different type of said antibody (Abd) - to said Ag bound to said solid carrier (SC^) ;
wherein:
(i) each type of said L-Abc and Abd-SC]_ has a lower average affinity constant (K) for said Ag than each respective K of said L- Aba and said Abb-SC.
3. The improved immunoassay of claim 2 wherein said additional entity is said L-Abc.
4. The improved immunoassay of claim 2 wherein Aba, Abb, Abc, and Abd are each independently selected from a group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof.
5. The improved immunoassay of claim 2 wherein Ab, and Abc are monoclonal antibodies.
6. The improved immunoassay of claim .4 wherein said additional entity is said L-Abc.
7. The improved immunoassay of claim 1 wherein said immunoassay is a two-step. sandwich immunoassay of the type comprising:
(a) contacting said fluid with said antibody (Abb) bound to a solid carrier (SC) to form an insoluble complex (SC-Ab -Ag);
(b) contacting said SC-Abb "-Ag with a soluble labeled antibody complex (SC-Ab -Ag-Aba-L) ;
(c) separating said SC-Abb-Ag-Aba-L from unreacted L-Aba; and
(d) measuring either the amount of L-Aba associated with .said SC-Ab -Ag-Aba-L or the amount of unreacted L-Aba; and is characterized in that in step (b) said SC-Abb-Ag is contacted with at least one different type of soluble, labeled antibody (L-Abc); wherein:
(i) each type of said L-Abc has a lower average affinity- constant (K) for said Ag . than the K of said L-Aba.
8. The improved immunoassay of claim 6 wherein Aba, Abb, and Abd are each independently selected from a group consisting of monoclonal antibodies, poly¬ clonal antibodies, and mixtures thereof.
9. The improved immunoassay of claim 7 wherein Aba and Abc are monoclonal antibodies.
10. The improved immunoassay of claim 1 wherein said immunoassay is a direct nephelometric immunoassay of the type comprising:
(a) contacting said fluid with said antibody (Ab) to said Ag in order to form a complex Ab-Ag; and
(b) measuring the amount of formed Ab-Ag; and is characterized in that said fluid is contacted with at least one additional type of antibody Abe, each type of said Abe having an average affinity constant (K) for said Ag lower than the K of said Ab for said Ag.
11. The improved direct nephelometric immunoassay of claim 10 wherein each additional type of Abe is a monoclonal antibody.
12. The improved direct nephelometric immunoassay of claim 16 wherein each additional type of Abe is a polyclonal antibody.
13. An improved reagent of the type comprising at least one first entity selected from a group consisting of
(a) an antibody (Ab) to an antigenic substance (Ag);
(b) a soluble, labeled antibody (L-Aba) to said Ag; and (c) an antibody (Abb) bound to a solid carrier (SC) , characterized in that said reagent further comprises at least one additional entity selected from a group consisting of at least one different type of soluble labeled antibody (L-Abc), at least one different type of antibody (Abd) to said Ag bound to a solid carrier (SC-^), and at least one different type of antibody (Abe) to said Ag, wherein:
(i) each of said additional entities has a lower average affinity constant (K) for said Ag than the K of its corresponding first entity; (ii) said additional entity is present in an amount sufficient to avoid a hook effect when said reagent is employed in an immunoassay; and (iii) said SC]_ is selected from a group consist¬ ing of said SC, at least one different solid carrier (SC2), an mixtures thereof (SC and SC ) •
14. The improved reagent of claim 13 being of the type comprising:
(a) said L-Aba to said Ag; and
(b) said Abb bound to said SC; and being characterized in that said reagent further comprises at least one additional entity selected from a group consisting of at least one different type of soluble labeled antibody (L-Abc) to said Ag and.at least one different type of antibody (Abd) to said Ag bound to a solid carrier (SC^); wherein:
(i) each type of said L-Abc and said Ab^-SC^ has a lower K for said Ag than each respective K of said 'L-Aba and said Ab - SC.
15. The improved reagent of claim 14 comprising:
(a) a first unit comprising said L-Aba to said Ag (and said L-Abc to said Ag)χ; and
(b) a second unit comprising said Abb~SC (and said Abd-SC1)y; where x and y are 0 or 1, provided that .when x and y are equal, x and y are 1.
16. The improved reagent of claim 15 wherein x is 1 and y is 0.
17. The improved reagent of claim 7 wherein said Aba, Ab , Abc, and Abd are each independently selected from a group consisting of monoclonal antibodies, polyclonal antibodies, and- mixtures thereof.
OMPI
18. The improved reagent of claim 17 wherein said Aba and Abc are monoclonal antibodies.
19. The improved reagent of claim 13 being of the type comprising said Ab, and being characterized in that said reagent further comprises at least one differ¬ ent type of Abe, each different type of said Abe having a K for said Ag lower than the K of said Ab for said Ag.
20. The improved reagent of claim 19 wherein each of said Abe is independently selected from a group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof.
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US4595661A (en) 1986-06-17
JPS60501776A (en) 1985-10-17

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